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. 2019 Jan 21;9(3):661–675. doi: 10.7150/thno.27794

Figure 4.

Figure 4

786-O cells knocked-out for VEGFC show increased proliferation and migration. (A) VEGFC mRNA levels were tested by qPCR in control (CTL) and two independent clones (Cl1, Cl2) knocked-out for VEGFC. (B) VEGFC protein levels were evaluated by ELISA in the supernatant of control (CTL) and two independent clones (Cl1, Cl2) knocked-out for VEGFC. (C) The locus of VEGFC was sequenced in control (CTL) and two independent clones (Cl1, Cl2) knocked-out for VEGFC; single or multi-base deletions were detected in the VEGFC locus. (D-E) The proliferation of control (CTL) or knock-out (Cl1, Cl2) cells were determined using clonogenic assays (D) or by quantification of the number of live cells (E). (F) Increased proliferation correlated with enhanced AKT activity evaluated by measuring the ratio of amount of phosphorylated to total AKT (pAKT/AKT). (G-H) VEGFC knock-out cells showed increased migration in Boyden chambers (G). (H) Quantification of the results shown in (G). (I) VEGFC knock-out cells showed increased levels of mesenchymal markers (Slug, Twist) detected by immuno-blotting; tubulin is shown as a loading control. Quantification of protein levels (mean ± SEM) is shown. Slug and Twist expressions in control cells (CTL) were considered reference values (100%); * p <0.05. When indicated, results are represented as the mean of at least three independent experiments ± SEM. ** p<0.01, *** p<0.001.